The ultimate goal of this project is to create a novel proteomic resource that will enable the real-time expression profiling of proteins in live murine embryonic stem (mES) cells and in differentiated cells derived therefrom. The resource will be in the form of an annotated bank of protein-trap mES cell lines, each of which will carry a single tagged gene. Fluorescent signal in these lines will report the expression and subcellular localization of the protein product(s) of the tagged gene in each line. Such a library of clonal fluorescently- tagged lines can be used, in conjunction with quantitative fluorescence microscopy in multiwell live-cell arrays, to study thousands of proteins in live cells, in real tim, under various experimental conditions. In a separate effort, we have initiated the development of such a bank of fluorescent protein-trap mES cell lines. However, the technology we have used so far will only allow us to detect and isolate protein-traps in genes that are actively expressed in mES cells; this limits the breadth and versatility of the library because the bulk of the proteome (>80%) is estimated to be effectively silent in mES cells. In order to create a truly broad proteomic library, we have devised a method that will allow us to identify and isolate protein-traps in genes that are silent in mES cells. Further, in this new approach, we will also add a more versatile protein tag (FAP-tag) so as to greatly increase the sensitivity of fluorescence detection in (and therefore the overall functionality of) the protein-trap mES library This Phase I project is designed to construct the requisite DNA vectors, to test their efficacy at identifying protein-traps in silent genes, and to assess their scope and suitability for use in a future large-scale protein-trapping effort.